The invention will occasionally be mentioned in the text as HBX
Spectral analysis is a basic measuring method of properties—chemical or physical as well as others—in a fluid with substances as suspension and/or solution. Spectral photometry of light absorption in chosen ranges of wave lengths is a well established standard method for determination of substances in a fluid and/or the actual condition of the substances at the time of measurement. The method is commonly used within medical technology for the analysis of human blood.
The patent application will in the following be concentrated to and described as the fluid blood, especially human blood for medical purpose, even though the invention is suitable for other body fluids or other organic/non organic fluids with or without substances in suspension, where the technical conditions makes the method applicable.
Blood is commonly described as a complex red fluid consisting of one bright yellow part, plasma, and in the plasma a suspension of blood cells mainly red cells. In an adult the blood volume is approximately 5 litre, of which 40-50% is red cells. The ratio between red cells and plasma is called hematocrit.
Plasma consists of mainly water with proteins, sugar, vitamins, hormones enzymes etcetera. The blood cells are mainly divided in three groups, red cells erythrocytes, with a size significant of 0.007 mm and normally in an amount of 4-5 million/mm3 of whole blood. AN erythrocyte consists of a thin membrane as a kind of balloon in which there is water and a high concentration of the protein haemoglobin (Hb) in various forms, substances that can bind to and release oxygen and carbon dioxide in the circulation.
Normally there is approx. 13-15 g haemoglobin per 100 ml blood in an adult, corresponding to 4-5 million red cell. Even a very small sample of blood taken from one person is a representative amount of blood cells for making measurements to determine certain properties and conditions of the blood.
The haemoglobin value is a measurement of the oxygen transportation capacity of the blood to other tissue and a parameter for patient diagnosis. Haemoglobin value is furthermore a primary safety and quality parameter in blood banking where blood is stored for transfusion purpose and collected as raw material for blood based industrial purposes.
Many other substances e.g. glucose in plasma, is measured by use of photometry as Hb. In general measurement of volumes, activity and condition for substances in blood is a basic diagnostic aid for determination of a persons/patients medical condition.
Haemoglobin (Hb) measurement is one of the most common diagnostic tests in the world today. Billions of tests are performed by use of different methods from the most elementary based on copper sulphate, to complete blood cell counts using sophisticated haematology analysers. These kind of tests are measured on a majority of the world's population one or several times during a life time. Considering the population of the world today of 6 billion and growing, the need for a low cost, yet high quality point of care measurement of Hb and other parameters is very big.
Of all tests performed today a great deal should benefit from being done with higher demand for safety and precision. The limited use of photometric method corresponding to these demands is probably a matter of time and cost. A preferred test method suitable for mass production must be subject to continuous cost reducing product development within the frames of both maintained quality and easiness of use also under rough conditions. Test methods must also fulfil stipulated specifications in standards.
Basically and not including so called non invasive measuring methods related to Hb, the cost of a Hb-test consists of the following factors exclusive the sampling:                time of measurement        cost of material/disposables        equipment, (purchase, handling, service, calibration, lifetime etc.        In addition to this is the cost for sampling which is basically the same for all capillary methods which is cheaper than venous/arterial blood sampling in tubes.        
The major users of single Hb-tests are the Transfusion Centres where the Red Cross is the biggest actor country wise. It is mandatory in most countries to check the Hb-value prior to donation. Another area where it is also mandatory in several countries to check for Hb-value is maternity care.
The best and most common technique for single Hb-measurement is by use of photometry. Normally light with specified wavelength passes through a small chamber (cuvette) containing the specimen. The cuvette can be of different size and shape and are often specially designed. The blood absorbs part of the light and the transmitting light is measured and the Hb-value calculated and displayed in a stable and precise way.
Haemoglobin determination can be made in different ways. Hb-fractions can be measured directly by use of multiple wave lengths or all haemoglobin can be measured indirectly by use of one or two wave lengths after being converted chemically to a stable colour complex e.g. acidmetemoglobin. Common for these methods is that measurement takes place after hemolysation, break down of the red cell membrane, creating a solution of haemoglobin.
It is possible to measure haemoglobin without haemolysing the red cells. This measurement can be done momentarily, which saves time and facilitates the handling, however brings great technical challenges. Whole blood with intact red cells is a colloid suspension with a strong tendency to scatter light, which if not handled in a right way will seriously disturb the measurement. Great consideration must be taken to minimize the scattering effects.
Indirect measurement of total Hb is used In most of the common systems of the less complex kind on the market today. By use of a cuvette that is prepared with reagent for hemolysation and transforming of the haemoglobin to a stable colour complex in typical one minute, thus avoiding the scattering effect but at the same time no specification of the different Hb-fractions. A system utilizing this method successfully has been on the market for more than 20 years and other almost identical systems from Germany and USA have been introduced to the market lately
A more complex and sophisticated method is developed and patented in USA. Based on direct measuring of haemoglobin fractions, so called CO-Oximetry in unaltered whole blood. The method is based on multiple wave length measuring of whole blood where the scattering effect is minimized by use of a large sensor in the photometer, collecting also scattered light. Algorithms are used to calculate and distinguish the Hb-fractions. Larger and more expensive disposable cuvettes are used in this system which is in terms of sophistication and price is less suitable for the mass market.
All together the above mentioned and on the market existing systems has good features but also some disadvantages that can be overcome with new technique, new components and new thinking. The disadvantages are relatively high cost per test, time consuming and shelf life of cuvettes with reagent as salt with sensitivity to moisture and thereby risk of incorrect measurements.
Microcuvettes are small cuvettes, size one to a few centimeter square. The cuvette, a type of container for liquids, is placed in an arrangement holding it in place for light penetration and measurement of transmitting light. The cuvette has a specific lumen in shape of a slit where the blood sample is placed and penetrated by light. Substances e.g. Hb-components have specific characteristics regarding light absorption/transmission, deflexion, refraction index etc. These characteristics are known for different Hb-components for example Hb bound to oxygen. Consequently the transmitted light is characterized by the absorption emanating from the substances in the sample together with the reflection and scattering that occurs when light is colliding with particles e.g. cells in during its passage through the sample. It is mainly the absorption that is the information searched in the transmitting light and—in the case of Haemoglobin—is presented in relation to the chosen He-components, for example oxygen saturation.
Each chosen wave length contributes with information and gives additional information about a certain Hb-fraction. Wave length of special interest is absorption max respective min and so called isobestic points. The totally transmitted light consists of a complex and the effect of scattering makes the interpretation of the measurement result even more complex.
In HBX a new way is chosen for the elimination of unwanted scattering effects, different from previous hemolysation or use of a big sensor for optimising incoming light. The blood is unaltered during the measurement. This is of importance since it is also possible to measure continuously in a blood stream without destroying and the measurement takes only a second. The problem of light scattering from the red cells effecting the measurement is solved through an optic-geometric design of the light conducting components and cuvette minimizing scattered light to reach the detector. Described more in detail below.
HBX is also based on a new combination of information searched, chosen light source, cuvette type, choice of spectrophotometer, measuring procedure, and signal handling optimised for the purpose and mathematical algorithms.
The invention comprises tree major parts, each one described below and in enclosed figures.
Optical Geometry
The solution of the scattering problem in the invention is based upon a fundamental theory for light scattering (reference Twersky, V. Absorption and multiple scattering by biological suspensions. J. Opt. Soc. Am. 60:1084-1093, 1970), in this case for fluids with suspended substance. The from theory emanating alternatives is determined by the actual conditions at the chosen optical/geometrical procedure. The design is correlated to the amount of scattered light to reach the detector of the photometer.
By arranging the light pathway to and from the cuvette using chosen angels of the light it is possible to prevent scattered light to reach the detector of the photometer or reduce the scattered light to a minimum and calculable level.
Optimal effect is reached by minimizing the measuring area to a size related to the size of the light pathway at the same time as the possible light deviation from said light pathway to and from the measuring object to the photometer is strongly limited. In principle this technique is designed to be contrary to the above mentioned USA method where a maximum of scattered light is collected for processing.
By choosing new fixed light sources with optimised characteristics e.g. light temperature, intensity (energy level) and adjustable exposure time or light sources with variable wave lengths it is possible to optimise the measuring set up for the spectrophotometer.
Basically two principles or alternatives are applicable in HBX; one is a broad band light source (white light) in connection to a spectrophotometer registration the light intensity as a function of wave length. The other alternative is a variable wave length mono chromatic light source of laser/maser in connection to a standard photometer registering absobrance/transmittant light intensity. Which alternative is chosen is determined by type of measuring object, situation and purpose.
The spectrophotometer in the first case is of type “monolithic multi-wavelengths diode-array, MMWDA” a new application (Hb-fractions) for this type of spectrophotometer for medical diagnostic purpose. The spectrophotometer is in this case combined with broad spectrum white light and measures the transmitting light from the light source at all wave lengths simultaneously.
The alternative combination is more like the previously existing standard methods with a simpler photometer in combination with a state of the art light source with variable frequency i.e. monochromatic light of different chosen wave lengths.
In prior art this was achieved by use of separate light sources, each with different wave length corrected by use of filters. In the alternative with MMWDA-photometer it is also possible but unnecessary to use a variable monochromatic light. Both combinations of light source and photometer separately makes it possible to directly measure and extract each of the searched haemoglobin fraction. This is one of the main characteristics of HBX which measures four different Hb-fractions The instrument is for the purpose equipped with reference values for all the actual Hb-fractions extinction curves. HBX admits the possibility to automatically present the value of each of the Hb-fractions and in addition the total Hb-value.
Signal Processing
Both of the above described alternatives light sources—photometer permits a great number of possible registrations of measuring data instantly, providing a possibility to choose freely the measuring points of interest from the complete measured sequences for further signal processing. Thereby the precision in the calculations can be decided and optimised.
The meted further admits calculation of the measuring error, for description of accuracy which is an integrated part of quality assurance.
The measurement is performed so quickly that no significant time for the measuring procedure needs to be taken into consideration. The complete analysis of all fractions is performed within a second which is unelectable in comparison with the other moments of an Hb-analysis e.g. sampling etc. This further admits consecutive measurements over cycles of 30-60 per minute or even continues measurements.
Signal processing involves processor, memory for reference and measured data, algorithms for the chosen measuring data and compensation for possible scattering effects or deviations emanating from abnormal/unexpected data. The algorithm for calculation/elimination of light scattering is based upon accepted theories described in scientific publications. Further algorithms and approximations are based on least square method.
The invention HBX comprise several part inventions in terms of:                1) An optical geometry for minimizing scattered light to the detector in order to make measurements possible for accurate determination of chosen substances in colloid solutions, for example whole blood.        2) Two alternative measuring principles with the same purpose of creating data for selection of measuring points over a wide wave length range. This can be based upon a firm broad band white light source in combination with a spectrophotometer or a variable narrow wave lengths light source in combination with a standard type photometer.        3) A series of algorithms for signal processing designed for standard corrections, for the optical geometry and for calculations of approximations by use of a number of chosen measuring points.        4) A combination of above described part solutions enables very fast measuring cycles. The time consuming for each measuring cycle permits measurements faster than for example heart rate which makes the method suitable for continuous measuring of blood flow.        
Even though HBX at every measuring situation enables a large amount of measuring data it not necessary to use all of these. In practise it is possible for a given purpose e.g. custom model/design of equipment, to utilize a limited or extended amount of data corresponding significantly to the purpose or application.